Abstract

Snake bite is crucial medical emergency while the success of immediate care and anti-venom therapy mainly depend on the correct identification of culprit snake. We present a simple method for identification of six snake species including Echiscoloratus, Echispyramidum, Cerastescerastesgaspreti, Bitisarieatans, Najahaje Arabica, andWalterinnesiaaegyptia, using the gel filtration chromatographic profiles of their venoms. The chromatograms of venoms from different snake species showed peculiar patterns based on the number and location of peaks.Our findings suggest that chromatographic profiles of snake venoms provide a simple and reproducible chemical fingerprinting method for quick identification of snake species.

Keywords

Introduction

The venoms from Elapidae and Viperidae snakes are
complex mixturescontaining different components such
asmetalloproteinases, proteolytic enzymes, phospholipase,
serine proteinase, presynaptic and postsynaptic neurotoxins,
potassium channel-binding neurotoxins, cytotoxins,
cardiotoxins and platelet aggregation inhibitors [1-3].Newton et al [4] have suggested that analysis of venom
components can produce a unique fingerprint to be used
as a valuable reference tool in taxonomic analysis asa
complementary method to morphology and behavioral
characterization for species identification and classification.
Compositionaldifferences between snake venoms
can be employed as a taxonomy signature forunambiguous
species identification independently of geographic
origin andmorphological characteristics [5]. Recently,
Calvete [6] have pointed out that use of proteomics approaches
in identification of evolutionary and immunoreactivitytrends
among homologous and heterologous venoms
may aid in the replacement of thetraditional geographic-
and phylogenetic-driven hypotheses for
antivenom production strategies.

In continuation to our previous work on chemical fingerprinting
of scorpion venoms [7], we performed the gel
filtration chromatography of snake venoms from four species from the family Viperidae (Echiscoloratus,
Echispyramidum, Cerastescerastesgaspreti and Bitisarieatans)
and two species from the family Elapidae (Najahaje
Arabica and Walterinnesiaaegyptia) and compared the
chromatographic profiles for their application in species
identification.

Materials and Methods

We collected six species of snakes (Fig. 1) including Echiscoloratus, Echispyramidum, Cerastescerastesgaspreti,
Bitisarieatans, Najahaje Arabica and Walterinnesiaaegyptiafrom the Riyadh region of Saudi Arabia.
The snakes were kept in plastic boxes and fed onmice and
water adlibitum. The crude venom was diluted with distilled
water, properly mixed, and centrifuged at 10,000
rpm at 4°C for 20 min to separate the mucus. The clear
supernatant was filtered through 0.20 μm filter before
chromatography.

Figure 1: Snake species used in this study.

Gelfiltration chromatography was used for venom fractionation
on Superdex200 PC 3.2/30 column. Venom solution
was diluted in 0.05 M sodium phosphate buffer
containing 0.15 M NaCl (pH 7.0). An aliquot (25μl) of
venom solution was loaded in a previously equilibrated
column with the same buffer. The sample was injected
using an Auto injector. The flow rate was adjusted at 0.4 mL/min and the UV range was 0.02 AUFS (absorbance
unit full scale). Column operational pressure was 1.5
MPa. The elution profile was monitored at 280 nm by a
UV spectrophotometer (AKTA Micro System). All the
samples were run in triplicate to confirm the reproducibility
of their chromatographic patterns.

Results and Discussion

All the snake species showed peculiar chromatographic
profiles of their venoms depending on the location and
height of the peaks (Fig 2). The minimum numbers of
peaks were observed with the venom of Cerastescerastesgaspreti (
3 peaks) and the maximum number of peaks
with the venom of Walterinnesiaaegyptia (7 peaks). Both
the members of the genus Echis (Echiscoloratus and Echispyramidum)showed 5 peaks each (3 common peaks)
whereas the venoms of Najahaje Arabica and Bitisarieatans resulted in 4 and 6 peaks respectively (Fig. 2).

The venom profile-based chemical fingerprinting clearly
differentiated the two species of the family Elapidae
(Naja Arabica and Walterinnesia Aegyptia) from the
members of the family Viperidae. The venom profile of Echiscoloratus was more closely related to the venom profile of Cerastesgaspreti instead of Echispyramidum (Fig. 2). Aird [8] compared the gel filtration profiles of
crude snake venoms from 38 Crotalusviridis,representing
the subspecies concolor, viridis and lutosus and readily
distinguished these taxa. John and Kaiser [9] conducted a
comparative study of the venoms from Notechisscutatusscutatus,
Notechisaterserventyi, Notechisaterhumphreysi
and Notechisateraterusing gel filtration resulting in
slightly different elution profiles on a Superose-12 gel
filtration column.The protein profile of venoms of Elapidae
was identified using electrofocusing technique; the
two species could easily be differentiated whereas the
differences between the two sub-species were more difficult
to evidence [10]. The elution profiles of the venoms
of seven Bothrops species fractionated on a Mono-Q
FPLC column resulted in reproducible chromatogramshowever
there was a considerable overlap of activeproteins
in different species venoms [11].

In conclusion, eachsnake’s venom has a unique chromatographic
profile that can be used as a fingerprint to differentiate
one speciesfrom the other.Our findings suggest
that gel filtration chromatography of snake venom is a
simple and reproducible method for identification of
snake species.

Acknowledgments

The authors extend their appreciation to the Deanship of
Scientific Research at King Saud University, Riyadh,
Saudi Arabia for funding the work through the research
group project No. RGP-VPP-009.